This invention relates to the field of cytogenetics. In particular it provides new diagnostic nucleic acid markers for prostate cancer.
Molecular genetic mechanisms responsible for the development and progression of prostate cancer remain largely unknown. Identification of sites of frequent and recurring allelic deletion or gain is a first step toward identifying some of the important genes involved in the malignant process. Previous studies in retinoblastoma (Friend, et al. Nature, 323:643-6 (1986)) and other cancers (Cawthon, et al., Cell, 62:193-201 (1990); Baker, et al., Science, 244:217-21 (1989); Shuin, et al., Cancer Res, 54:2832-5 (1994)) have amply demonstrated that definition of regional chromosomal deletions occurring in the genomes of human tumors can serve as useful diagnostic markers for disease and are an important initial step towards identification of critical genes. Similarly, regions of common chromosomal gain have been associated with amplification of specific genes (Visakorpi, et al., Nature Genetics, 9:401-6 (1995)). Additionally, definition of the full spectrum of common allelic changes in prostate cancer may lead to the association of specific changes with clinical outcome, as indicated by recent studies in colon cancer and Wilms"" tumor (Jen, et al., N. Engl. J. Med., 331:213-21 (1994); Grundy, et al., Cancer Res, 54:2331-3 (1994)).
Prostate cancer allelotyping studies (Carter, et al., Proc Natl Acad Sci USA, 87:8751-5 (1990); Kunimi, et al., Genomics, 11:530-6 (1991)) designed to investigate one or two loci on many chromosomal arms have revealed frequent loss of heterozygosity (LOH) on chromosomes 8p (50%), 10p (55%), 10q (30%), 16q (31-60%) and 18q (17-43%). Recently, several groups have performed more detailed deletion mapping studies in some of these regions. On 8p, the high frequency of allelic loss has been confirmed, and the regions of common deletion have been narrowed (Bova, et al., Cancer Res, 53:3869-73 (1993); MacGrogan, et al., Genes Chromosom Cancer, 10:151-159 (1994); Bergerheim, et al., Genes Chromosom Cancer, 3:215-20 (1991); Chang, et al., Am T Pathol, 144:1-6 (1994); Trapman, et al., Cancer Res, 54:6061-4 (1994); Suzuki, et al., Genes Chromosom Cancer, 13:168-74 (1995)). Similar efforts also served to narrow the region of common deletion on chromosome 16q (Bergerheim, et al., Genes Chromosom Cancer, 3:215-20 (1991); Cher, et al., J Urol, 153:249-54 (1995)). Other prostate cancer allelotyping studies utilizing a smaller number of polymorphic markers have not revealed new areas of interest (Phillips, et al., Br J Urol, 73:390-5 (1994); Sake, et al., Cancer Res, 54:3273-7 (1994); Latil, et al., Genes Chromosom Cancer, 11:119-25 (1994); Massenkeil, et al., Anticancer Res, 14:2785-90 (1994)). At present, allelotyping studies are limited by the low number of loci studied, low case numbers, heterogeneous groups of patients, the use of tumors of low or unclear purity, and lack of standardization of experimental techniques. For these reasons, it has been difficult to compare frequencies of alterations between studies, and we have yet to gain an overall view of regional chromosomal alterations occurring in this disease.
Comparative genomic hybridization (CGH) is a relatively new molecular technique used to screen DNA from tumors for regional chromosomal alterations (Kallioniemi, et al., Science, 258:818-21 (1992) and WO 93/18186). Unlike microsatellite or Southern analysis allelotyping studies, which typically sample far less than 0.1% of the total genome, a significant advantage of CGH is that all chromosome arms are scanned for losses and gains. Moreover, because CGH does not rely on naturally occurring polymorphisms, all regions are informative, whereas polymorphism-based techniques are limited by homozygous (uninformative) alleles among a fraction of tumors studied at every locus.
CGH can detect and map single copy losses and gains in prostate cancer with a high degree of accuracy when compared with the standard techniques of allelotyping (Cher, et al., Genes Chromosom Cancer, 11:153-162 (1994)). Copy-number karyotype maps have been generated for prostate cancer showing several recurrently altered regions of the genome (Cher, et al., Genes Chromosom Cancer, 11:153-162 (1994); Visakorpi, et al., Cancer Res, 55:342-347 (1995)).
Although previous studies have begun to reveal a genome-wide view of chromosomal alterations occurring in primary and recurrent prostate cancer, metastatic prostate cancer has not been examined in depth. The present invention addresses these and other needs in the prior art.
The present invention provides compositions and methods of detecting a genetic alterations correlated with prostate cancer. The methods comprise contacting a nucleic acid sample from a patient with a probe which binds selectively to a target polynucleotide sequence correlated with prostate cancer. The invention provides the following chromosomal regions which are deleted in prostate cancer cells: 2q, 4q, 5q, 6q, 10p, and 15q. Regions which show increases in copy number in prostate cancer cells are: 1q, 2p, 3q, 3p, 4q, 6p, 7p, 7q, 9q, 11p, 16p, and 17q.
The probes of the invention are contacted with the sample under conditions in which the probe binds selectively with the target polynucleotide sequence to form a hybridization complex. The formation of the hybridization complex is then detected.
Alternatively, sample DNA from the patient can be fluorescently labeled and competitively hybridized against fluorescently labeled normal DNA to normal lymphocyte metaphases. Alterations in DNA copy number in the sample DNA are then detected as increases or decreases in sample DNA as compared to normal DNA.
The chromosome abnormality is typically a deletion or an increase in copy number. The methods can be used to detect both metastatic prostate cancers and in androgen independent prostate cancer.
Definitions
A xe2x80x9cnucleic acid samplexe2x80x9d as used herein refers to a sample comprising DNA in a form suitable for hybridization to a probes of the invention. For instance, the nucleic acid sample can be a tissue or cell sample prepared for standard in situ hybridization methods described below. The sample is prepared such that individual chromosomes remain substantially intact and typically comprises metaphase spreads or interphase nuclei prepared according to standard techniques.
The sample may also be isolated nucleic acids immobilized on a solid surface (e.g., nitrocellulose) for use in Southern or dot blot hybridizations and the like. In some cases, the nucleic acids may be amplified using standard techniques such as PCR, prior to the hybridization. The sample is typically taken from a patient suspected of having a prostate cancer associated with the abnormality being detected. xe2x80x9cNucleic acidxe2x80x9d refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, would encompass known analogs of natural nucleotides that can function in a similar manner as naturally occurring nucleotides.
xe2x80x9cSubsequencexe2x80x9d refers to a sequence of nucleic acids that comprise a part of a longer sequence of nucleic acids.
A xe2x80x9cprobexe2x80x9d or a xe2x80x9cnucleic acid probexe2x80x9d, as used herein, is defined to be a collection of one or more nucleic acid fragments whose hybridization to a target can be detected. The probe is labeled as described below so that its binding to the target can be detected. The probe is produced from a source of nucleic acids from one or more particular (preselected) portions of the genome, for example one or more clones, an isolated whole chromosome or chromosome fragment, or a collection of polymerase chain reaction (PCR) amplification products. The probes of the present invention are produced from nucleic acids found in the regions of genetic alteration as described herein. The probe may be processed in some manner, for example, by blocking or removal of repetitive nucleic acids or enrichment with unique nucleic acids. Thus the word xe2x80x9cprobexe2x80x9d may be used herein to refer not only to the detectable nucleic acids, but to the detectable nucleic acids in the form in which they are applied to the target, for example, with the blocking nucleic acids, etc. The blocking nucleic acid may also be referred to separately. What xe2x80x9cprobexe2x80x9d refers to specifically is clear from the context in which the word is used.
xe2x80x9cHybridizingxe2x80x9d refers the binding of two single stranded nucleic acids via complementary base pairing.
xe2x80x9cBind(s) substantiallyxe2x80x9d or xe2x80x9cbinds specificallyxe2x80x9d or xe2x80x9cbinds selectivelyxe2x80x9d or xe2x80x9chybridizing specifically toxe2x80x9d refers to complementary hybridization between a probe and a target sequence and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target polynucleotide sequence. These terms also refer to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA. The term xe2x80x9cstringent conditionsxe2x80x9d refers to conditions under which a probe will hybridize to its target subsequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 5xc2x0 C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Typically, stringent conditions will be those in which the salt concentration is at least about 0.02 Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 60xc2x0 C. Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
One of skill will recognize that the precise sequence of the particular probes described herein can be modified to a certain degree to produce probes that are xe2x80x9csubstantially identicalxe2x80x9d to the disclosed probes, but retain the ability to bind substantially to the target sequences. Such modifications are specifically covered by reference to the individual probes herein. The term xe2x80x9csubstantial identityxe2x80x9d of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 90% sequence identity, more preferably at least 95%, compared to a reference sequence using the methods described below using standard parameters.
Two nucleic acid sequences are said to be xe2x80x9cidenticalxe2x80x9d if the sequence of nucleotides in the two sequences is the same when aligned for maximum correspondence as described below. The term xe2x80x9ccomplementary toxe2x80x9d is used herein to mean that the complementary sequence is identical to all or a portion of a reference polynucleotide sequence.
Sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two sequences over a xe2x80x9ccomparison windowxe2x80x9d to identify and compare local regions of sequence similarity. A xe2x80x9ccomparison windowxe2x80x9d, as used herein, refers to a segment of at least about 20 contiguous positions, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444 (1988), by computerized implementations of these algorithms.
xe2x80x9cPercentage of sequence identityxe2x80x9d is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to the same sequence under stringent conditions. Stringent conditions are sequence dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 5xc2x0 C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Typically, stringent conditions will be those as described above.